1. Field of the Invention
This invention relates to an accelerator of the activity of hydrolase, especially one that accelerates the activation of the precursors of serine protease, including blood coagulation Factor XII, and that accelerates the enzyme activity of serine protease produced by the activation of the said precursors. Moreover, this invention relates to an accelerator of blood coagulation with the use of the said accelerator.
2. Description of the Prior Art
The remarkable progress in testing techniques coupled with the spreading of blood tests including serum biochemistry tests, serum immunology tests, and blood cell tests is contributing much to the prevention of disease and to early diagnosis. Most blood tests are tests of the serum, and the serum that is needed for such tests is, as a rule, obtained by blood that is collected into a container for blood tests and allowed to coagulate, after which centrifugation takes place, by which the blood clot (the gel-like mass of a mixture of fibrin and blood cells), which has a different specific gravity from that of the serum, is separated therefrom, and the serum is collected with the use of a pipette or by decantation.
A relatively long period of time is needed for blood collected from subjects to be tested to coagulate. For example, even if glass test vessels, which allow blood to coagulate in a relatively short period of time, are used, it takes 40-60 minutes until coagulation, and if test vessels made of a synthetic resin are used, the vessels must be left for 4 hours or more. For that reason, there is the problem that it is not possible to obtain serum needed for tests speedily. This is a particular problem when the test is to be done in a emergency.
A blood coagulation accelerator is used so that blood will coagulate rapidly. Factor XII is one form of precursor of a protein hydrolase (serine protease) related to blood coagulation, and by its activation, other blood coagulation factors in the blood are activated in turn to start blood coagulation. As accelerators of the activity of Factor XII, conventionally, glass, kaolin, bentonite, silica, and other inorganic fine or coloidal particles, and ellagic acid are known. These are also used as one ingredient among the reagents for the measurement of the activated partial thromboplastin time (APTT), which is one kind of test of coagulation functioning. However, even if these are used as blood-coagulation accelerators, differences in their purity and composition cause scattering of the results for tests such as the time for blood coagulation. In addition, even if these accelerators of blood coagulation are used to promote coagulation of the blood, the separation effect of the blood serum and the blood clot is incomplete, so when centrifugation is done to obtain the serum, some components of the blood clot are mixed into the serum, which is a disadvantage.
A further problem arises when serum is obtained from the blood of patients who receive hemodialysis or patients with blood-clotting disorders. Such patients are given heparin to prevent the formation of clots, so 1-20 units of heparin is found in every 10 ml of their blood. This heparin binds with antithrombin III in their blood and strongly inhibits the effects of thrombin. It is said that the heparin also inhibits the effects of blood coagulation factors including Factor XII. For that reason, fibrinogen does not become fibrin, and so blood does not coagulate. Even if the accelerators of blood coagulation mentioned above are added, the blood does not actually coagulate, and so it is difficult to obtain the serum.
The inventors of this invention have proposed in Japanese Laid Open Patent Publication No. 60-115519 that an organic cyclic compound with the following general formula has carbonyl groups adjacent to each other in the structure that are substantially in the same plane as each other and is useful for accelerators of the activity of hydrolase, which can be used for accelerators of blood coagulation. ##STR1## (wherein A is the cyclic compound moiety.)
As above mentioned compounds, for example, there are oxidized alkyl gallate, oxidized ellagic acid, etc. These compounds non-enzymatically activate blood coagulation Factor XII, and make it possible for the blood to coagulate in a relatively short period of time. The effect is stronger than that obtained with the use of conventional accelerators of blood coagulation. The inventors have also proposed an accelerator of blood coagulation in which these compounds are the main ingredient, or in which various additives are added to these compounds. For example, applications have already been filed for an accelerator of blood coagulation that contains protein hydrolase as a co-accelerator to promote further the coagulation of the blood (Japanese Laid Open Patent Publication No. 60-174952) and for an accelerator of blood coagulation that includes a neutralizer of heparin so that the heparin in the blood of patients receiving heparin will be neutralized (Japanese Laid Open Patent Publication No. 60-27858).
It is possible to use all kinds of proteases including trypsin, thrombin, and cathepsin B as protein hydrolases for the acceleration of blood coagulation. These protein hydrolases act to accelerate the activation of blood coagulation factors in the blood, so the blood coagulates rapidly. For the neutralizer of heparin mentioned above, compounds with amine salts and/or a quaternary nitrogen can be used, including alkylamine hydrochloride. With an accelerator of blood coagulation that includes such compounds, the above-mentioned amine salts, etc., adsorb onto heparin, neutralizing it, and act to inactivate the heparin; moreover, the compounds with the structure of formula I activate blood coagulation Factor XII in the blood, so that it becomes possible for the blood to coagulate in a short period of time.
As mentioned above, by the use of an accelerator of blood coagulation containing compounds of the structure shown in formula I as the main ingredient, it is now possible to obtain serum from blood that has coagulated in a short period of time, having been taken from either healthy persons or patients given heparin. There is not a large scattering in the time taken for the blood to coagulate. However, the compounds of the structure shown in formula I mutually interact with some components of blood and influence the data obtained by clinical examinations, so that the use of the compounds as the main ingredient of an accelerator of blood coagulation must be limited.